Certain fungal diseases are significant economic problems with regard to some crop species. One of the class of fungi which are pathogens of many crops belong to the Verticillium genus of fungal pathogens which includes two economically important plant pathogens, Verticillium dahliae and Verticillium albo-atrum. Verticillium dahliae is a particularly problematic pathogen for potato. In current agronomic practices, farmers raising potatoes are forced to soil fumigants to control the presence of Verticillium dahliae. Otherwise, farmers run the risk that large portions of their crop will be lost to an early dying disease caused by the fungus. However, Verticillium dahliae is not present in all of the fields in which potatoes are grown. Even in infected fields, the level of infection is not always significant. Typically, in current practice, the soil fumigation is applied to the fields in which potato is to be grown whether or not Verticillium dahliae is present, just to avoid a catastrophic loss of the crops. The soil fumigation process is, however, quite expensive and is environmentally undesirable. It is therefore desirable to diagnose in advance whether or not Verticillium dahliae is present in the fields in which potatoes are to be grown, so as to determine whether or not soil fumigation is necessary in order to safely cultivate potatoes in the field.
Existing protocols for diagnosis of Verticillium species rely on in vitro culture of soil samples using selective media. The selective media are, at least in theory, selective for the presence of the particular species Verticillium dahliae. However, existing tests report large numbers of false negative responses. In addition, the tests are laborious and take three to four weeks to perform, which can be an unsatisfactory time period for agricultural purposes. Accordingly, a faster and more accurate diagnostic test for the presence of Verticillium dahliae would be useful in commercial potato growing.
One method that is developed as a general assay for biological systems is based on the polymerase chain reaction referred to as PCR. Using PCR, it is possible to make many copies, in a process referred to as amplification, of a desired DNA sequence if, and only if, the sequence is present in an environmental or other specimen. Thus, if a DNA sequence is known which is diagnostic of a particular species or condition, it becomes possible to perform a PCR analysis on an environmental sample, amplifying the specific DNA in question, and thus be able to determine whether or not the organism in question was present in that sample.
The PCR process is keyed by primers, which are specific to a DNA sequence to be amplified. PCR primers have been developed which allow for detection of Verticillium fungi. However, the previously known differences between V. dahliae and V. albo-atrum are based on a single, or very few, base pair differences. Thus, a PCR assay based on those few differences requires very stringent conditions to be effective. Prior to the method described here, no known larger DNA sequence was diagnostic of Verticillium dahliae and capable of distinguishing it from V. albo-atrum.